PROJECT SUMMARY This is an R35 application in response to RFA-GM-17-002 ?Maximizing Investigators? Research Award (R35). Our laboratory focuses on studying the pleiotropic second messenger cyclic AMP (cAMP) system. cAMP is a major stress signal that regulates a myriad of important biological processes under both physiological and pathological conditions, including cancer, chronic pain, diabetes, heart failure, and infections. Hence, not surprisingly, the cAMP signaling cascade is one of the most targeted pathways by current pharmaceuticals. In multi-cellular eukaryotic organisms, the effects of cAMP are mainly transduced by two ubiquitously-expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the more recently discovered exchange proteins directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF). While accumulating evidence implicates EPAC proteins as important stress response switches in the development of various human diseases, major gaps in our basic understanding of EPAC structures and functions persist. To bridge these gaps, we will apply structural, pharmacological and genetic approaches to interrogate the mechanisms of action and biological functions of this important family of signaling molecules, leading to the assessment of EPAC proteins as potential therapeutic targets in various disease models. The proposed research is based on more than seventeen years of extensive fundamental studies of EPAC-mediated signaling and directly builds on a several recent developments in our laboratory, which include the characterization of EPAC knockout mice and the discovery of first-in-class EPAC specific inhibitors. The combination of complementary structural approaches, novel genetic animal models and pharmacological probes will enable us to reveal much desired mechanistic insight and in vivo functions of EPAC proteins. This research plan will also aid in the advancement of new pharmacological tools for deciphering EPAC-mediated cell signaling and disease mechanisms, which can pave the way for novel, mechanism-based therapeutic strategies specifically targeting cAMP/EPAC signaling.